Process for the preparation of β-lactam derivatives

ABSTRACT

A process for the preparation of Cefuroxime acid (I), which comprises the following steps: (1) Extraction of deacetyl 7-glutaryl ACA (II) aqueous solution at acid pH with organic solvents (for example according to the procedures disclosed in U.S. Pat. No. 5,801,241); (2) drying the resulting solution while preventing lactonization of the intermediate; (3) carbamoylation of the hydroxymethyl group at the 3-position by reaction with chlorosulfonyl isocyanate or similar products; (7) extraction of the carbamoyl derivative from step 3 with water at neutral pH; (8) enzymatic hydrolysis of the amide at the 7-position of the cephalosporanic ring with glutaryl acylase; (6) acylation of the amnino group by condensation with 2-furanyl(sin-methoxyimino)acetic acid chloride or mixed anhydride.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a National Stage entry of International ApplicationNo. PCT/EP00/04372, filed May 16, 2000, the entire specification claimsand drawings of which are incorporated herewith by reference.

The present invention generally relates to the field of organicchemistry.

More particularly, the invention relates to a process for thepreparation of Cefuroxime acid, i.e.(6R,7R)-7-[[2-furanyl(sin-methoxyimino)acetyl]amino]-3-carbamoyloxymethylceph-3-em-4-carboxylic)acid, and the salts thereof, starting from(6R,7R)-7-[(4-carboxy-1-oxobutyl)amino]-3-hydroxymethyl-ceph-3-em-4-carboxylicacid (deacetyl 7-glutaryl ACA).

Cefuroxime acid is a key intermediate for the industrial synthesis oftwo third generation cephalosporins, Cefuroxime sodium (for theinjection administration) and Cefuroxime Axetil (for the oraladministration). These molecules are therapeutically valuable thanks totheir effective broad spectrum antibacterial activity againstgram-negative bacterials, in particular in the treatment ofimmunodepressed patients. Their effectiveness is advantageously combinedwith remarkable resistance to βlactamases.

The synthesis of Cefuroxime disclosed in U.S. Pat. No. 3,966,717 andU.S. Pat. No. 3,974,153 comprises 8 synthetic steps starting from 7-ACA(7-amino cephalosporanic acid). Such high number of steps, which causesa low overall yield, is due to the introduction of two protectivegroups, the first (e.g. thienyl acetyl) on the amine function and thesecond (e.g. benzhydryl) on the 7-ACA acid function.

Subsequently, processes starting from 7-ACA have been developed (Wilson,E.M. Chemistry and Industry 1984, 217) which do not involve the use ofprotective groups and remarkably reduce the number of steps. Inparticular, the best process, illustrated in Scheme 1, comprises 3steps:

1. Conversion of 7-ACA into deacetyl-7-ACA;

2. Acylation of the amino group;

3. Carbamoylation of the C-3 alcohol group (Scheme 1).

It has now surprisingly been found that Cefuroxime can be preparedstarting from intermediates of 7-ACA enzymatic synthesis withoutisolating any intermediate.

The enzymatic synthesis of 7-ACA involves, depending on the usedprocess, an intermediate that can either be glutaryl-7-ACA (U.S. Pat.No. 5,424,196; Bianchi, D., Bortolo, R., Golini, P., Cesti, P. LaChimica e l'Industria 1998, 80, 879), which can be enzymaticallyconverted into deacetyl 7-glutaryl ACA (II), or (II) itself, fromfermentative processes yielding des-Cephalosporin C (U.S. Pat. No.4,533,632).

The process object of this invention, illustrated in Scheme 2, comprisesthe following steps:

1. Extraction of deacetyl 7-glutaryl ACA (II) aqueous solution at acidpH with organic solvents (for example according to the proceduresdisclosed in U.S. Pat. No. 5,801,241).

2. Drying the resulting solution while preventing lactonization of theintermediate.

3. Carbamoylation of the hydroxymethyl group at the 3-position byreaction with chlorosulfonyl isocyanate or similar products.

4. Extraction of the carbamoyl derivative from step 3 with water atneutral pH.

5. Enzymatic hydrolysis of the amide at the 7-position of thecephalosporanic ring with glutaryl acylase.

6. Acylation of the amino group by condensation with2-furanyl(sin-methoxyimino)acetic acid chloride or mixed anhydride.

The process of the invention involves a reduction of the number of thesteps compared with the known processes for the preparation ofCefuroxime, as it requires neither the protection of the carboxyl at the4-position of the cephalosporanic ring nor that of the amino group atthe 7-position nor the recovery of any intermediates, thus causing aremarkable increase in the overall yield directly starting fromdes-Cephalosporin C or Cephalosporin C fermentation broth afterenzymatic deacetylation.

Furthermore, the process of the invention allows making use ofintermediates, which, contrary to 7-ACA, are particularly stable inaqueous solution.

The process according to the present invention provides Cefuroxime acidor a salt thereof which can be transformed into the correspondingcommercial products Cefuroxime sodium and Cefuroxime axetil.

The process described above comprises the preparation of anintermediate, which has to day never been described, namely(6R,7R)-7-[(4-carboxy-1-oxobutyl)amino]-3-carbamoyloxy-methyl-ceph-3-em-4-carboxylicacid of formula (III)

or a salt thereof.

The steps of the process according to the present invention for thepreparation of Cefuroxime acid starting from deacetyl 7-glutaryl ACA(II) are described in detail hereinbelow.

1. Extraction of a deacetyl 7-glutaryl ACA (II) aqueous solution with anorganic solvent, preferably cyclohexanone (U.S. Pat. No. 5,801,241).

A deacetyl 7-glutaryl ACA aqueous solution, at a concentration rangingfrom 1 to 20%, is adjusted to pH ranging from 1.0 to 3.0, preferably1.5, at temperatures ranging from 0 to 15° C., preferably from 0 to 5°C. These conditions prevent degradation of the substrate to give alactone following condensation of the carboxyl at the 4-position withthe hydroxyl bound to the methyl at the 3-position. The resultingsolution is added with 0.5 to 2 volumes of an organic solvent,preferably cyclohexanone, and extraction is carried out at temperaturesranging from 0 to 5° C. Phase are separated, then the aqueous phase isback-extracted with 0.5÷1.0 volumes of solvent and the organic phasesare combined.

2. The resulting organic solution is brought to temperatures from 0 to15° C., preferably from 0 to 5° C., adjusted to apparent pH ranging from6 to 8, preferably 7, with a solution of triethylamine in the organicsolvent used for the extraction. The resulting mixture is concentratedin vacuo and at a temperature below 25° C., to obtain a suspension witha water content below 0.5%, which is then processed in the subsequentstep.

3. Conversion of deacetyl 7-glutaryl ACA (II) into the corresponding3-carbamoyloxymethyl derivative (III), by reaction in cyclohexanone withan activated isocyanate, preferably chlorosulfonyl isocyanate. Thesuspension isolated at the end of the previous step, having aconcentration ranging from 1 to 10%, is cooled to temperatures rangingfrom −30 to 0° C., preferably −10° C., and added, in small portions,with 1÷5 mols of chlorosulfonyl isocyanate per mol of substrate. Theresulting heterogeneous mixture is then kept at such temperature untilcompletion of the reaction, whose progression is monitored by HPLCchromatography.

4. Extraction of the carbamoyl derivative obtained in step 3. Thesolution from step 3 is added with 0.1÷0.3 volumes of cold water and theresulting heterogeneous mixture is then adjusted to pH ranging from 6 to8, preferably 7, at temperatures ranging from 0 to 15° C., preferablyfrom 0 to 5° C., with an aqueous ammonia solution. The two phases areseparated; the organic phase is back-extracted with water, to 0.2volumes; the aqueous phases are combined and the resulting solution,having a concentration ranging from 5 to 30%, is processed in thesubsequent step.

5. Conversion of the 7-glutaryl 3-carbamoyloxymethyl derivative (III)into the corresponding 7-β-amino derivative (IV) by enzymatic hydrolysisof the amide at the 7-position of the cephalosporanic ring with glutarylacylase. The resulting solution from the previous step is added with aglutaryl acylase isolated from an Escherichia Coli culture, suitablysupported on a macroreticular resin, preferably polyacrylic epoxide, andthe resulting suspension is kept at pH 7.0-9.0, preferably 7.5, attemperatures ranging from 20 to 30° C., preferably 25° C., untilcompletion of the reaction. The progression of the hydrolysis of theglutaryl derivative to the corresponding cephalosporanic ring ismonitored by HPLC chromatography. The reaction yield is higher than 85%.After completion of the reaction, the enzyme is removed and the productis converted into Cefuroxime acid.

6. (6R,7R)-7-Amino-3-carbamoyloxymethylceph-3-em-4-carboxylic acid (IV)is directly converted into Cefuroxime acid (I), adding the aqueoussolution with a concentrated methylene chloride solution of2-furanyl(sin-methoxyimino)-acetic acid chloride or mixed anhydride(preferably the chloride).

Optionally, intermediate (IV) can be recovered by acidification toisoelectric pH of the solution from the previous step, then be worked inthe acylation of the amino group at the 7-position of thecephalosporanic ring, by following the method described in example 2 ofU.S. Pat. No. 3,974,153.

EXAMPLE 1

300 ml of a 6% deacetyl 7-glutaryl ACA aqueous solution, cooled to 0÷5°C., is added with the necessary amount of sulfuric acid to adjust pH to1.5. The resulting solution is extracted with one volume ofcyclohexanone. The organic phase, kept at a 0÷5° C., is added with thetriethylamine amount necessary to adjust pH to 6.0. The solution is thenconcentrated to water content below 0.5. At the end of the operation,350 ml of a glutaryl deacetyl 7-ACA suspension containing 98% of theactivity present in the solution resulting from extraction at acid pH,are obtained. The resulting solution is then transformed in thesubsequent step.

EXAMPLE 2

The mixture from the previous step is cooled to −10IC and then addedwith 13 ml of chlorosulfonyl isocyanate, preventing temperature fromexceeding −10° C. After completion of the addition of the reactive, themixture is kept at −10° C. until the starting product disappears. At theend of the synthesis, the reaction yield is checked to be about 95%. 150ml of cold water are added. The heterogeneous mixture is kept at a 0÷5°and its pH is adjusted with a 10% sodium carbonate solution. The twophases are separated, the organic phase is back-extracted with 50 ml ofwater. The resulting solution is then worked in the subsequent step.

The product from the above step was recovered adjusting to pH=2 thefinal aqueous solution saturated with sodium chloride. The isolatedrecovered was then characterized by analysis of the data from spectraMass and NMR spectrometry.

¹H-NMR: 1.82 ppm, qui, J=7 Hz, 2H, side chain CH₂; 2.3 ppm, m, 4H, sidechain methylenes; AB system at 3.4 ppm, methylene at the 2-position;system at 2.3 ppm, methylene bound to the oxygen; 5.05 ppm, d, J=5 Hz,1H, H-6; 5.55 ppm, d, J=5 Hz, 1H, H-7. ¹³C-NMR: 22.81 ppm (CH₂); 25.92ppm (CH₂); 35.61 ppm (CH₂); 36.99 ppm (CH₂); 57.98 ppm (CH); 58.90 ppm(CH); 65.84 ppm (CH₂—OR); 117.14 ppm (C-2); 132.04 ppm (C-3); 154.69 ppm(—OCON—); 165.78 ppm (two amide C═O); 169.30 ppm (COO—); 177.73 ppm(COOH). Electrospray ESI: m/z 388; m/z 410; m/z 344; m/z 327; m/z 349;m/z 309; m/z 299; m/z 281; m/z 253; m/z 185; m/z 172. Desadsorptionchemical ionization (DCI): m/z 233; m/z 214; m/z 216

EXAMPLE 3

The resulting aqueous solution is diluted with 300 ml of water and addedwith 40 g of glutaryl 7-ACA acylase, whose preparation is described inexample 12 of U.S. Pat. No. 5,424,196. The heterogeneous mixture stirredat 20° C. until completion of the reaction, automatically adjusting pHto 7.5 with 5% ammonia. The maximum conversion takes place after 60′,when the solution yield is about 86%. The enzyme is removed and theresulting solution is processed in the subsequent step.

EXAMPLE 4

A mixture of phosphorous pentachloride (10 g) and methylene chloride(100 ml), in a reactor of suitable volume, cooled at 0° C., is addedwith 18.4 ml of N,N-dimethylacetamide. The resulting solution is cooled,added with of 8.5 g of 2-furanyl(sin-methoxyimino)acetic acid andstirred at −10° C. for 15′; 22 ml of cold water are added thereto,keeping the resulting heterogeneous mixture stirred, the two phases areseparated and the organic phase is slowly added to the solution of thesubstrate isolated in the previous step (example 3), suitably cooled,automatically adjusting pH with 10% sodium hydroxide. The resultingmixture is kept at 0° C. until the substrate disappears. The finalmixture is added with 50 ml of N,N-dimethylacetamide, 50 ml ofacetonitrile and 300 ml of water, pH is adjusted to 2 with 2Nhydrochloric acid and the resulting mixture is stirred at 0÷5° C. forone hour, filtered, washed with water, and dried under-vacuum at 30÷35°C. for 8 hours. 15 g of Cefuroxime acid are obtained in an 85% yield.

EXAMPLE 5

The product from the step described in example 3 is isolated byadjusting to the isoelectric pH the aqueous solution obtained at the endof this step.

A mixture of 8.4 g of said product, i.e. of (6R,7R)-7-amino-3-carbomoiloxymethylceph-3-em-4-carboxylic (IV) acid, 37 mlof N,N-dimethylacetamide, 37 ml of acetonitrile, 21 ml of triethylamineand 5 ml of water is stirred at 0÷2° C. until complete dissolution ofthe product. Separately, a suspension of 7.7 g of phosphorouspentachloride in 75 ml of methylene chloride is added at 0÷2° C. with14.2 ml of N,N-dimethylacetamide and the resulting solution is added at−10° C. with 6.5 g of 2-furanyl(sin-methoxyimino)acetic acid; theresulting solution is added with 17 ml of cold water, keeping themixture at 0÷2° C. for about 15′. After that, the two phases areseparated and the organic phase is added to the substrate solutionprepared above, suitably cooled at −5° C. The reaction mixture is keptat 5÷10° C. until the substrate disappears. The final mixture is addedat 0÷5° C. to a mixture of 570 ml of water and 50 ml of 2N hydrochloricacid, pH is adjusted to=2 with 2N hydrochloric acid and the resultingmixture is stirred at 0÷5° C. for about an hour, then filtered, washedwith water and dried at 30÷35° C. for 8 hours. 11 g, of Cefuroxime acidare obtained in an 86% yield.

What is claimed is:
 1. A process for the preparation of Cefuroxime acid(I)

starting from deacetyl 7-glutaryl ACA (II), which comprises thefollowing steps, without recovery of any intermediates: a) extracting adeacetyl 7-glutaryl ACA (II) aqueous solution at pH 1-3 and at atemperature of 0-15° C. with an organic solvent, to obtain an organicphase containing deacetyl 7-glutaryl ACA (II)

b) adjusting said organic phase to pH 6-8, then drying such phase bydistillation under vacuum at a temperature below or equal to 25° C.; c)reacting deacetyl 7-glutaryl ACA (II) with an activated isocyanate attemperatures ranging from −30 to 0° C., to obtain(6R,7R)-7-[(4-carboxy-1-oxobutyl)amino]-3-carbamoyloxymethyl-ceph-3-em-4-carboxylicacid (III)

d) extracting said compound (III) from the reaction mixture with waterat pH of 6-8 and at a temperature of 0-15° C.; e) transforming compound(III) into (6R,7R)-7-amino-3-carbamoyloxymethyl-ceph-3-em-4-carboxylicacid (IV)

by glutaryl acylase enzymatic hydrolysis, at pH 7-9 and at a temperatureof 20-30° C.; f) condensing compound (IV) with a2-furanyl(sin-methoxyimino)acetic acid halide or anhydride, to obtainthe desired Cefuroxime acid (I).
 2. A process as claimed in claim 1, inwhich said organic solvent is cyclohexanone.
 3. A process as claimed inclaim 2, in which said deacetyl 7-glutaryl ACA (II) aqueous solution hasa concentration of 1-20%.
 4. A process as claimed in claim 1, in whichin step b) the organic phase is dried to a water content below 0.5%. 5.A process as claimed in claim 1, in which said activated isocyanate ischlorosulfonyl isocyanate.
 6. A process as claimed in claim 1, in whichsaid glutaryl acylase is supported on a macroreticular resin.
 7. Aprocess as claimed in claim 6, in which said resin is a polyacrylicepoxide resin.
 8. A process as claimed in claim 7, in which saidglutaryl acylase is isolated from an Escherichia Coli culture.